Agarose gel electrophoresis is a widely used laboratory technique that separates DNA or RNA fragments based on their size. This method involves moving charged molecules through a porous agarose matrix under an electric field. Voltage influences both the speed and the quality of the nucleic acid separation.
Principles of Voltage in Electrophoresis
DNA molecules inherently possess a negative charge. When an electric current is applied, these negatively charged DNA molecules are drawn towards the positively charged electrode, or anode. The gel acts as a sieve, a porous matrix through which DNA must navigate. Smaller DNA fragments move through these pores more easily and migrate faster and farther than larger fragments. A higher applied voltage creates a stronger electric field, causing them to migrate more quickly through the gel. This relationship between voltage and migration speed is generally proportional at lower voltage settings, typically below 5 volts per centimeter (V/cm).
Key Factors for Voltage Selection
Selecting the appropriate voltage is important for optimal results in agarose gel electrophoresis. Several factors influence this choice, including gel concentration, DNA fragment size, buffer type and ionic strength, and desired run time. Power supply capabilities can also play a role.
Gel concentration is a primary consideration, as it dictates the pore size within the agarose matrix. Higher percentage gels have smaller pores, which slow DNA migration and are better suited for separating smaller DNA fragments. Conversely, lower percentage gels have larger pores, allowing for better separation of larger DNA molecules. For optimal resolution, lower voltages are generally used with higher percentage gels, while higher voltages can be applied to lower percentage gels. For instance, a 0.8% gel provides good separation for 5–10 kilobase pair (kb) DNA fragments, whereas a 2% gel is suitable for 0.2–1 kb fragments.
The size of the DNA fragments to be separated also directly influences voltage choice. To resolve smaller fragments, a higher voltage might be considered to accelerate their movement, but this must be balanced with resolution. For larger DNA fragments, lower voltages are often preferred to ensure they migrate adequately through the gel and separate well. For optimal resolution of DNA greater than 2 kb, a voltage of 5 to 8 V/cm (distance between electrodes) is commonly suggested for standard gel electrophoresis.
The type and ionic strength of the buffer solution used are also significant factors. Buffers like Tris-acetate-EDTA (TAE) and Tris-borate-EDTA (TBE) are commonly employed, each with different conductivities. TAE buffer generally has higher conductivity, allowing DNA to move quickly, useful for routine separations. TBE buffer, with its lower conductivity, generates less heat, which can lead to sharper bands and better resolution, particularly for fragments smaller than 2 kb. The ionic composition and pH of the running buffer affect the migration of charged particles.
Finally, the desired run time involves a trade-off between speed and resolution. Higher voltages can significantly reduce the time required for a run, but this can negatively impact the clarity of the separated bands. For maximum resolution, particularly of larger DNA fragments, running the gel at a lower voltage for a longer duration is often recommended. Some large agarose gels may be run overnight at very low voltages, such as 1-1.5 V/cm, for fine separation.
Impact of Voltage on Results
The chosen voltage setting has a direct and observable impact on the quality of the electrophoresis results. When the voltage is set appropriately, the outcome is typically clear, well-separated bands, indicating good resolution of the DNA fragments. This allows for accurate assessment of fragment sizes and quantities.
However, using a voltage that is too high can lead to several problems. Excessive voltage causes increased heat generation within the gel. This heat can lead to the gel melting or distorting, especially with low melting point agarose gels. High heat can also result in “smiling” bands, where the center of the DNA bands migrates faster than the edges, creating a curved appearance. This uneven migration is due to temperature variations across the gel, with the center heating up more than the edges. High voltage can decrease resolution, leading to band streaking for larger DNA molecules and potentially causing DNA denaturation.
Conversely, setting the voltage too low also has disadvantages. A very low voltage results in significantly slower migration times for the DNA fragments. This extended run time can cause band diffusion, where the DNA spreads out and the bands become fuzzy or less distinct. In some cases, separation might be incomplete within a practical timeframe, making it difficult to analyze the results. While lower voltage can improve resolution by reducing heat, the trade-off is often a prolonged experiment duration. Finding a balance between achieving good resolution and efficient run time is a consistent consideration in agarose gel electrophoresis.